In the animal kingdom, nonvascular plants, fungi, yeast and bacteria, the primary reserve polysaccharide is glycogen. Glycogen is a D-glucose polysaccharide containing linear molecules with .alpha.-1,4 glycosyl linkages and is branched via .alpha.-1,6 glycosyl linkages. Although glycogen is analogous to starch from a linkage comparison, glycogen exhibits a different chain length and degree of polymerization. In bacteria, for example, the .alpha.-1,6 glycosyl linkages constitute only approximately 10% of the total linkages, indicating that the majority of the glycogen polymer resides as linear glucose units.
In vascular plants, reserve polysaccharides are stored in roots, tubers and seeds in the form of starch. Starch, a complex polymer of D-glucose, consists of a mixture of linear chain (amylose) and branched chain (amylopectin) glucans. Starches isolated from different plants are found to have distinct proportions of amylose. Typically, amylose comprises from about 10-25% of plant starch, the remainder being the branched polymer amylopectin. Amylopectin contains low molecular weight chains and high molecular weight chains, with the low molecular weight chains ranging from 5-30 glucose units and the high molecular weight chains from 30-100 or more. The ratio of amylose/amylopectin and the distribution of low molecular weight to high molecular weight chains in the amylopectin fraction are known to affect the properties, such as thermal stabilization, retrogradation, and viscosity, and therefore the utility of starch. The highest published low m.w./high m.w. chain ratios (on a weight basis) in amylopectin are 3.9/1 for waxy corn starch, which has unique properties. Additionally, duwx, which has slightly more branch points than waxy, also has further unique properties.
In addition, starches from different plants or plant parts often have different properties. For example, potato starch has different properties than other starches, some of which may be due to the presence of phosphate groups. In some plant species, mutants have been identified which have altered contents of amylose and amylopectin. Mutations that affect the activity of starch-branching enzyme in peas, for example, result in seeds having less starch and a lower proportion of amylopectin. Also, mutations in the waxy locus of maize, which encodes a starch granule bound starch synthase, result in plants which produce amylopectin exclusively. Similarly, a potato mutant has been identified whose starch is amylose-free (Hovenkamp-Hermelink et al. Theor. Appl. Genet. (1987) 75:217-221). It has been found that varying the degree of starch branching can confer desirable physical properties; other changes in the characteristics of native starch could result in the production of polymers with new applications.
Cyclodextrins are the products of enzymatic starch degradation by a class of amylases termed cyclodextrin glycosyltransferase (CGT) enzymes. The family of cyclodextrins contains three major and several minor cyclic oligosaccharides which are composed of a number of homogenous cyclic .alpha.-1,4-linked glucopyranose units. The cyclodextrin having six glucopyranose units is termed .alpha.-cyclodextrin (also know as Schardinger's .alpha.-dextrin, cyclomaltohexaose, cyclohexaglucan, cyclohexaamylose, .alpha.-CD, ACD and C6A). The seven unit cyclodextrin is termed .beta.-cyclodextrin (also known as Schardinger's .beta.-dextrin, cyclomaltoheptaose, cycloheptaglucan, .beta.-CD, BCD and C7A). The eight unit cyclodextrin is termed .gamma.-cyclodextrin (also known as Schardinger's .gamma.-dextrin, cyclomaltooctaose, cyclooctaglucan, cyclooctaamylose, .gamma.-CD, GCD and C8A).
The cyclic nature of cyclodextrins allows them to function as clathrates (inclusion complexes) in which a guest molecule is enclosed in the hydrophobic cavity of the cyclodextrin host without resort to primary valence forces. Thus, the components are bound as a consequence of geometric factors, and the presence of one component does not significantly affect the structure of the other component. Complexing a hydrophobic compound with cyclodextrin increases the stability and solubility of the hydrophobic compound. Applications of this phenomena have been found in many fields including pharmaceuticals, foods cosmetics and pesticides.
In pharmaceutical applications, complexing a drug with cyclodextrins for oral delivery can have many advantages. Among the benefits are the transformation of liquids into solids which can be formed into tablets, stabilization of drugs against volatilization and oxidation, reduction of bad taste or smell, improvement in the rate of dissolution of poorly soluble drugs and increases in blood levels of poorly water soluble drugs (Pitha, in Controlled Drug Delivery, Bruck, ed. Vol. 1, p. 125, (1983) CRC Press). From the limited research done on parenteral administration of cyclodextrin-complexed drugs, some of the same advantages found for oral delivery can also be observed. The undesirable side effects of drugs can be reduced with complexation with cyclodextrins. Such side affects include gastric irritation from oral delivery, local irritation and hemorrhagic areas from intramuscular injection, and local irritation from eye-drops (Szejtli, J., Cyclodextrin Technology, Kluwer Academic Publications, Boston (1988), pp. 186-306).
The addition of cyclodextrins to food products or cosmetics can also have many effects. In spices, food flavoring or perfume fragrances, cyclodextrins protect against oxidation, volatility, and degradation by heat or light (Hashimoto, H., "Application of Cyclodextrins to Food, Toiletries and Other Products in Japan," in Proceedings of the Fourth International Symposium of Cyclodextrins, O. Huber and J. Szejtli, eds. (1988) pp. 533-543). Cyclodextrins can also eliminate or reduce undesirable smells or tastes, and modify food or cosmetic textures.
Complexing pesticides with cyclodextrins can increase the bioavailability of poorly wettable or slightly soluble substances, and transform volatile liquids or sublimable solids into stable solid powders (Szejtli, J. (1988) supra at pp. 335-364; U.S. Pat. No. 4,923,853). Pesticides which are sensitive to light, heat or oxygen degradation can be stabilized by complexing with cyclodextrins.
Currently, production of cyclodextrins begins with the cultivation of an appropriate microorganism, e.g., Bacillus macerans, and separation, purification and concentration of the amylase enzyme. The enzyme is then used to convert a starch substrate to a mixture of cyclic and acyclic dextrins. Subsequent separation and purification of cyclodextrins is then required. The bacterial strain from which the enzyme is isolated and the length of time the starch conversion is allowed to progress determines the predominant form of cyclodextrin produced. Manufactures of .alpha.-cyclodextrins attempt to manipulate the reaction to preferentially make the specific cyclodextrin, however, the process is not easily controlled, and a mixture of cyclodextrins is obtained. At the present time .beta.-cyclodextrin is the most widely commercialized form of cyclodextrin because the .beta.-form is much cheaper to produce than the .alpha.- or .gamma.-cyclodextrins.
In 1987, the U.S. market for cyclodextrins was predicted to reach $50 million per year within 2 years; that figure would double if the U.S. Food and Drug Administration approved the use of cyclodextrins in food (Seltzer, R., Chem. Eng. News, (May 1987) pp. 24-25). The world market is estimated to be twice the U.S. figure (Szejtli, J. (1988) supra at p. viii). The potential U.S. market for cyclodextrins has been predicted to reach as high as $245 million per year (Anon., Bioproc. Technol., Nov. 1987). There is potentially a large market waiting to be tapped if the cost of cyclodextrins could be lowered through alternative production methods.
With the development of genetic engineering techniques, it is now possible to transfer genes from a variety of organism into the genome of a large number of different plant species. This process is preferable to plant breeding techniques whereby genes can only be transferred from one plant in a species to another plant in the same or a closely related species. It would thus be desirable to develop plant varieties through genetic engineering, which have increased capacity for starch synthesis, altered amylose/amylopectin ratios, altered distribution of low to high molecular weight chains in the amylopectin fraction and also starches with novel molecular weight characteristics. In this manner, useful starches with a variety of viscosity or texture differences may be obtained.
In addition, recognizing the disadvantages of bacterial-derived CGT-mediated cyclodextrin production, it is considered desirable to produce cyclodextrins where CGT is the expression product of a recombinant gene transferred into a plant host. In this method, generically known as molecular farming, plants are transformed with a structural gene of interest and the product extracted and purified from a harvested field of the transgenic plants. For example, human serum albumin has been produced in transgenic tobacco and potato (Sijmons, P. C. et al., Bio/Technology (1990) 8:217-221).
Extending the idea of molecular farming to cyclodextrins provides a means to lower production costs. One particularly desirable host plant for such transformation is potato because of the large amount of starch production in potato tubers. A typical tuber contains approximately 16% of its fresh weight as starch (Burton, W. G., The Potato (1966) 3rd Edition, Longman Scientific and Technical Publications, England, p. 361). Transformation of potato plants with the bacterial CGT structural gene linked to a tuber-specific promoter and a leader directing the enzyme, for example, to the amyloplast, provides a means to produce large quantities of cyclodextrins in tubers.
To this end, nucleic acid sequences which encode glycogen biosynthetic or degradative enzymes are desirable for study and manipulation of the starch biosynthetic pathway. In particular, these enzymes may be expressed in plant cells using plant genetic engineering techniques and targeted to a plastid where starch synthesis occurs. It was therefore considered desirable to apply recombinant deoxyribonucleic acid (rDNA) and related technologies to provide for modified reserve polysaccharides in transgenic plants.
Proceeding from the seminal work of Cohen & Boyer, U.S. Pat. No. 4,237,224, rDNA technology has become available to provide novel DNA sequences and to produce heterologous proteins in transformed cell cultures. In general, the joining of DNA from different organisms relies on the excision of DNA sequences using restriction endonucleases. These enzymes are used to cut donor DNA at very specific locations, resulting in gene fragments which contain the DNA sequences of interest. Alternatively, structural genes coding for desired peptides and regulatory control sequences of interest can now be produced synthetically to form such DNA fragments.
These DNA fragments usually contain short single-stranded tails at each end, termed "sticky-ends". These sticky-ended fragments can then be ligated to complementary fragments in expression vehicles which have been prepared, e.g., by digestion with the same restriction endonucleases. Having created an expression vector which contains the structural gene of interest in proper orientation with the control elements, one can use this vector to transform host cells and express the desired gene product with the cellular machinery available. Recombinant DNA technology provides the opportunity for modifying plants to allow the expression of desirable enzymes in planta.
However, while the general methods are easy to summarize, the construction of an expression vector containing a desired structural gene is a difficult process and the successful expression of the desired gene product in significant amounts while retaining its biological activity is not readily predictable. Frequently, bacterial-derived gene products are not biologically active when expressed in plant systems.
To successfully modify plants using rDNA, one must usually modify the naturally occurring plant cell in a manner in which the cell can be used to generate a plant which retains the modification. Even in successful cases, it is often essential that the modification be subject to regulation. That is, it is desirable that the particular gene be regulated as to the differentiation of the cells and maturation of the plant tissue. In the case of glycogen synthase, ADP-glucose pyrophosphorylase and/or cyclodextrin glycosyltransferase, it is also important that the modification be performed at a site where the product will be directed to contact the reserve polysaccharide regions of the modified plant. Thus, genetic engineering of plants with rDNA presents substantially increased degrees of difficulty.
In addition, the need to regenerate plants from the modified cells greatly extends the period of time before one can establish the utility of the genetic construct. It is also important to establish that the particular constructs will be useful in a variety of different plant species. Furthermore, one may wish to localize the expression of the particular construct in specific sites and it is desirable that the genetically modified plant retain the modification through a number of generations.
Relevant Literature
The structural genes encoding the E. coli glycogen biosynthetic enzymes have been cloned (Okita, et al. (1981) J. Biol. Chem. 256:6944-6952) and their nucleic acid sequences determined (Preiss, J. (1984) Ann. Rev. Microbiol. 38:419-458; Kumar et al. (1986) J. Biol. Chem. 261:16256-16259). Genes encoding mammalian glycogen synthases have also been cloned and their nucleic acid sequences determined (Browner, et al. Proc. Nat. Acad. Sci. (1989) 86:1443-1447; Bai, et al., J. Biol Chem. (1990) 265:7843-7848).